Molecular characterizations of type IIb sodium dependent phosphate cotransporter in mouse intestine.

摘要

Inorganic phosphate (P_i) homeostasis is mainly regulated by absorption of dietary P_i in the small intestine and reabsorption of filtered P_i in the kidney. I have characterized the 5 ′-flanking region and overall gene structure of the murine type IIb sodium-phosphate cotransporter (Na/P_i-IIb), a major apical P_i transporter. The Na/P_i-IIb cotransporter gene spans more than 18 kb and consists of 12 introns and 13 exons. Three promoter/reporter gene constructs, −159/+73, −429/+73 and −954/+73, showed significant luciferase activity when transfected into in rat intestinal epithelial (RIE-1) cells.; P_i requirement during development is much higher than in adult life. In the next set of experiments, I sought to characterize expression of the intestinal Na/P_i-IIb cotransporter during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na/P_i uptake by intestinal brush-border membrane vesicles was highest at 14-days-of-age, lower at 21 days and further reduced at 8 weeks and 8–9 months of age. Na/P_i-IIb mRNA and immunoreactive protein levels in 14-d animals were markedly higher than in older groups. MP treatment significantly decreased Na/P_i uptake, and Na/P _i-IIb mRNA and protein expression in 14-d mice. Additionally, the size of the protein was smaller in 14-d mice. Deglycosylation of protein from 14-d and 8-wk old animals with PNGase F reduced the molecular weight to the predicted size. I conclude that intestinal Na/P_i uptake and Na/P_i-IIb expression are highest at 14-d and decrease with age. Furthermore, MP treatment reduced intestinal Na/P_i uptake ∼3-fold in 14-d mice and this reduction correlates with reduced Na/P_i-IIb mRNA and protein expression. I also demonstrate that Na/P_i-IIb is an N-linked glycoprotein and that glycosylation is age-dependent.; In conclusion, the mouse intestinal Na/P_i-IIb cotransporter is developmentally regulated at mRNA and protein levels. MP-treatment also reduces mRNA and protein expression during development. The Na/P_i-IIb gene promoter constructs identified in the first study will be a useful tool to investigate the possible transcriptional regulations. Furthermore, studying post-translational regulation including glycosylation will reveal developmental effects on Na/P_i-IIb cotransporter protein. These studies will help to decipher molecular mechanisms of P_i absorption in mammalian small intestine.